This MIRA proposal consolidates funded research on three central problems in cell biology concerning membrane dynamics: synaptic vesicle exocytosis, dynamic membrane contacts, and synthetic biology-enabled tests of Golgi models. Synaptic vesicles fuse to release neurotransmitters in less than 1 msec after Ca++ enters, enabling effective information processing in the brain. In spite of all of the progress in our understanding of membrane fusion generally, it is remarkable that we have no clear explanation of this. Our goal for the next 5-10 years is to develop a detailed structural biochemical and biophysical understanding of how neurotransmitter release is coupled to Ca++ and how it can occur so very rapidly. Two related problems must be solved to provide an answer: how is the half-zippered SNARE complex stabilized (clamped) from completing fusion spontaneously? How can fusion be completed 100-1000 times faster than permitted by the physical chemistry of individual SNAREs? Our general hypothesis is that sub-millisecond exocytosis is achieved in some way by a supra-molecular assembly involving the Ca++ sensor synaptotagmin and the clamp/activator complexin that harnesses and synchronizes the force of many SNAREs co-operatively to enable explosive release. Membrane contacts generally are dynamic entities, forming and breaking as the result of signals and shifts in physiology. The Golgi stack is based on such membrane contacts, and despite the fact that it looks like a stable entity, our recent and surprising results reveal that the principal proteins responsible for registered stacking (GRASP proteins) cycle on and off the membrane every minute! Nanoscopy-based imaging of suggests this is due to rapid cycles of fatty acid (palmitate) addition and cleavage in specialized transien adhesive nano-domains. Over the next 5 years we hope to elucidate the mechanisms involved using real-time nanoscopy of the enzymes, substrates and their products in living cells combined with detailed biochemical, biophysical, and genetic studies. The Golgi apparatus is found in all eukaryotes, and yet has the dubious and remarkable distinction of being the last remaining membrane organelle whose basic principle of operation is still unknown! In spite of decades of intensive research we still debate how proteins traffic across the Golgi stack in animal cells, simply because we lack the ability to directly observe protein transfer reactions in living cells at sufficient spatial/temporal resolution. The new idea we are developing is to engineer the conversion of the animal Golgi stack into a more yeast-like unstacked topology in living cells, so that we can now much more easily track individual cisternae, vesicles and tubules in relation to transport cargo and machinery, especially with the help of dynamic nanoscopy now possible in our department.